Polynucleotides encoding proteins mediating switch recombination

ABSTRACT

The present invention provides isolated SRTA-70 proteins that mediate immunoglobulin class switch recombination in antibody-producing cells, and methods of procuding such proteins. The invention further provides isolated polynucleotides encoding SRTA-70 proteins, as well as vectors and host cells comprising the polynucleotides. The invention further provides methods of using SRTA-70 proteins to identify agents that modulate immunoglobulin class switch.

The present invention relates to the isolation, purification andcharacterization of proteins mediating switch recombination. The presentinvention further relates to the microbial production via recombinantDNA technology of recombination protein SRTA-70, a member of theproteins mediating switch recombination. The present invention furtherrelates to the use of these proteins as therapeutically active agents inimmune response modulation, specifically, in augmentation andsuppression of the immune response.

Higher eukaryotes produce immunoglobulins (Ig) of diffent classes, whichare defined by the constant region (C) of the heavy (H) chain. Uponstimulation by antigen expression of the early IgM class changes to thatof another H chain class. This switch from one H chain class to another,named simply “class switching”, occurs via DNA recombination. Switchrecombination imprecisely joins two so-called switch (S) regions, whichlie upstream of the H chain genes and contain highly repetitivesequences (for reviews see Esser and Radbruch, Annu. Rev. Immunol. 8,717-735 [1990] and Harriman et al., Annu. Rev. Immunol. 11, 361-384[1993]). The recombination mechanism for most class switching events canbe described by the loop-excision model (Jäck et al., Proc. Natl. Acad.Sci. USA 85, 1581-1585 [1988]). The biochemistry of the class switchrecombination process, however, remains largely unknown.

In order to study the mechanism of class switch recombination an assaythat measures DNA-transfer activity was devised which makes use of two S(Sμ and Sγ2b) regions, cloned into two different, largely non-homologousvectors (FIG. 1). Using this assay three proteins in the S-RegionTransfer Activity (SRTA) were identified: B23 (nucleophosmin), poly(ADP) ribose polymerase (PARP) and a novel 70-KDa protein SRTA-70.

Thus, in a first aspect of this invention, there are provided SRTA-70proteins, specifically recombinantly produced SRTA-70 protein. The term“recombinantly produced SRTA-70 protein” refers to the protein of SEQ IDNo. 1 or any protein or polypeptide having an amino acid sequence whichis substantially homologous to the amino acid sequence SEQ ID No. 1 andfurther having the biological activities of the protein of SEQ ID No. 1.

As used hereinbefore the term “substantially homologous” means that aparticular subject sequence, for example, a mutant sequence, varies froma reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.For purposes of the present invention, sequences having greater than 95percent homology, equivalent biological activity and equivalentexpression characteristics are considered substantially homologous. Forpurposes of determining homology, truncation of the sequence should bedisregarded. Sequences having lesser degrees of homology, comparablebioactivity, and equivalent expression characteristics, e.g., fragmentsof the amino acid sequence SEQ ID No: 1 are considered substantialequivalents.

As used herein the term recombinantly produced SRTA-70 protein includesproteins modified deliberately, as for example, by addition of specificsequences that preferably bind to an affinity carrier material. Examplesof such sequences are sequences containing at least two adjacenthistidine residues (see in this respect European Patent No. 282 042).Such sequences bind selectively to nitrilotriacetic acid nickel chelateresins (Hochuli and Döbeli, Biol. Chem. Hoope-Seyler 368, 748 [1987];European Patent No. 253 303). SRTA-70 proteins which contain such aspecific sequence can, therefore, be separated selectively from theremaining polypeptides. The specific sequence can be linked either tothe C-terminus or the N-terminus of the SRTA-70 protein.

There are further provided isolated DNA sequences encoding SRTA-70proteins or fragments thereof. Specifically, the DNA sequences of thisinvention are defined to include the nucleotide sequence SEQ ID No: 2 ora fragment thereof or any DNA sequence which is substantially homologousto the nucleotide sequence SEQ ID No: 2 or a fragment thereof.

As used hereinbefore the term “substantially homologous”, means that aparticular subject sequence, for example, a mutant sequence, varies froma reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.For purposes of the present invention, DNA sequences having greater than95 percent homology, encoding equivalent biological properties, andshowing equivalent expression characteristics are consideredsubstantially homologous. For purposes of determining homology,truncation of the DNA sequence should be disregarded. Sequences havinglesser degrees of homology, encoding comparable bioactivity, and showingequivalent expression characteristics, e.g., fragments of the nucleotidesequence SEQ ID No: 2 are considered substantial equivalents. Generally,homologous DNA sequences can be identified by cross-hybridization understandard hybridization conditions of moderate stringency.

There are also provided vectors and expression vectors containing theDNA sequences of the present invention, hosts containing such vectorsfor the production of SRTA-70 proteins, and processes for the productionof such DNA sequences, recombinant vectors and host cells.

Methods for the expression, isolation and purification of the SRTA-70proteins are also provided.

The following steps outline the methods for recombinantly expressing theSRTA-70 proteins.

1) Cloning of DNA Sequences Encoding SRTA-70 Proteins

DNA sequences encoding SRTA-70 proteins can be cloned using a variety oftechniques. Using the methods described in this application cDNAsencoding SRTA-70 proteins or fragments thereof can be produced. ThesecDNAs can be isolated and amplified by PCR technique usingoligodeoxynucleotide DNA primers by conventional techniques.

The cDNA (SEQ ID No: 2) encoding the amino acid sequence SEQ ID No:1 isobtained using the DNA primers described in the examples. By usingconventional technique, this cDNA has been isolated from a mouse spleencDNA library.

The cDNA may be obtained not only from cDNA libraries, but by otherconventional techniques, e.g., by cloning genomic DNA, or fragmentsthereof, purified from the desired cells. These procedures are describedby Sambrook et al., in “DNA Cloning: A Practical Approach”, Vol. I andII, D. N. Glover, ed., 1985, MRL Press, Ltd., Oxford, U. K.; Benton andDavis, Science 196, 180-182 [1977]; Grunstein and Hogness, Proc. Nat.Acad. Sci. 72, 3961-3965 [1975]; and Maniatis et al., in “MolecularCloning-A Laboratory Manual”, Cold Spring Harbor Laboratory [1989].

To obtain the cDNA encoding the SRTA-70 proteins cDNA libraries arescreened by conventional DNA hybridization techniques by the methods ofBenton and Davis, supra, or Grunstein and Hogness, supra, usingradioactive SRTA-70 gene fragments. Clones which hybridize to theradioactive gene fragments are analyzed, e.g., by restrictionendonuclease cleavage or agarose gel electrophoresis. After isolatingseveral positive clones the positive insert of one clone is subcloned,e.g., into phagemids, and sequenced by conventional techniques.

Clones derived from genomic DNA may contain regulatory and intron DNAregions in addition to coding regions; clones derived from cDNA will notcontain intron sequences. In the molecular cloning of the gene fromgenomic DNA, DNA fragments are generated, some of which will encode thedesired gene. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNAse in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The linear DNA fragments can then beseparated according to size by standard techniques, including but notlimited to, agarose and polyacrylamide gel electrophoresis and columnchromatography.

Whatever the source, the DNA sequence encoding SRTA-70 proteins may bemolecularily cloned into a suitable vector for propagation of the DNA bymethods known in the art. Any commercially available vector may be used.For example, the DNA may be inserted into a pBluescript SK⁻ vector.Appropriate vectors for use with bacterial hosts are described byPouwels et al., in “Cloning Vectors: A Laboratory, Manual”, 1985,Elsevier, N.Y. As a representative but nonlimiting example, usefulcloning vectors for bacterial use can comprise a selectable marker and abacterial origin of replication derived from commercially availableplasmids which are in turn derived from the well known cloning vectorpBR322 (ATCC 37017). Such commercial vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (PromegaBiotec, Madison, Wis., USA).

The DNA sequences encoding SRTA-70 proteins inserted in thesecommercially available vectors can be verified by methods known in theart, e.g., by standard nucleotide sequencing techniques.

DNA sequences that code for SRTA-70 proteins from mammals other thanmice may be used herein. Accordingly, while specific DNA has been clonedand sequenced in relation to the DNA sequence in mouse cells, anymammalian or vertebrate cell potentially can be used as the nucleic acidsource of the SRTA-70 protein.

2) Production of SRTA-70 Proteins

Cloned DNA sequences that code for SRTA-70 proteins can be expressed inhosts to enable the production of these proteins with greaterefficiency. Techniques for these genetic manipulations are specific forthe different available hosts and are known in the art.

For expression of SRTA-70 proteins in hosts, in principle, all vectorswhich replicate and express DNA sequences encoding the SRTA-70 proteinsin the chosen host are suitable. Expression vectors suitable for use inprokaryotic host cells are mentioned, for example, in the textbooks“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory[1982] and [1989], of Maniatis et al. Examples of other vectors areplasmids of the pDS family (Bujard et al., Methods in Enzymology, eds.Wu and Grossmann, Academic Press, Inc., Vol. 155, 416-433 [1987]).

Such prokaryotic expression vectors which contain the DNA sequencescoding for the SRTA-70 proteins operatively linked with an expressioncontrol sequence can be incorporated using conventional methods into anysuitable prokaryotic host cell. The selection of a suitable prokaryotichost cell is determined by different factors which are well-known in theart. Thus, for example, compatibility with the chosen vector, toxicityof the expression product, expression characteristics, necessarybiological safety precautions and costs play a role and a compromisebetween all of these factors must be found.

Suitable prokaryotic host organisms include gram-negative andgram-positive bacteria, for example E. coli and B. subtilis strains.Examples of prokaryotic host organisms are E. coli strain M15 (describedas strain OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474[1974] and E. coli W3110 [ATCC No. 27325]). In addition to theaforementioned E. coli strains, however, other generally accessible E.coli strains such as E. coli 294 (ATCC No. 31446) and E. coli RR1 (ATCCNo. 31343) can also be used. In a preferred embodiment of the presentinvention E. coli M15 is used as the host organism.

Expression vectors suitable for use in yeast cells are described in“Guide to yeast genetics and molecular biology”, Guthrie and Fink, eds.,Methods in Enzymology, Academic Press, Inc., Vol. 194 (1991) and “Geneexpression technology”, Goeddel, ed., Methods in Enzymology, AcademicPress, Inc., Vol. 185 [1991]. Examples of suitable yeast cells areSaccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Schizosaccharomyces pombe cells. An overview on various yeast expressionsystems is given by Romanos et al., Yeast, Vol. 8, 423-488 [1992].

The transformation with the yeast expression vectors is carried out asdescribed by Klebe et al., Gene, Vol. 25, 333-341 [1983].

Plants can also be used as hosts for the production of SRTA-70 proteinof the present invention. Transfer of the DNA sequence coding for theSRTA-70 protein may be achieved by a variety of methods (for review seePotrykus and Spangenberg, eds., Gene transfer to plants. A laboratorymanual, Springer Verlag, Heidelberg, Germany [1995]), whereby the DNAsequence for the SRTA-70 protein is integrated into the chromosome ofthe host plants. Over-expression of the SRTA-70 protein may be achieved,for example, by transforming a plant host with the DNA sequence codingfor the SRTA-70 protein. Examples of plant hosts for the production ofSRTA-70 protein include, but are not limited to maize (Zea mays, Ishidaet al., Nature Biotechnology 14, 745-750 [1996]), flax (Linumusitatissimum, Dong and Mchughen, Plant Sci. 88 (1), 61-71 [1993]),soybean (Glycine max, Christou et al., Tibtech 8, 145-151 [1990]),alfalfa or tobacco.

The manner in which the expression of the SRTA-70 proteins is carriedout depends on the chosen expression vector host cell system.

Usually, the prokaryotic host cells which contain a desired expressionvector are grown under conditions which are optimal for the growth ofthe prokaryotic host cells. At the end of the exponential growth, whenthe increase in cell number per unit time decreases, the expression ofthe desired SRTA-70 protein is induced, i.e., the DNA coding for thedesired SRTA-70 protein is transcribed and the transcribed mRNA istranslated. The induction can be carried out by adding an inducer or aderepressor to the growth medium or by altering a physical parameter,e.g., a change in temperature. For example, the expression can becontrolled by the lac repressor.

By adding isopropyl-β-D-thiogalactopyranoside (IPTG), the expressioncontrol sequence is derepressed and the synthesis of the desired proteinis thereby induced.

The yeast host cells which contain a desired expression vector are grownunder conditions which are optimal for the growth of the yeast hostcells. A typical expression vector contains the promoter element, whichmediates the transcription of mRNA, the protein coding sequence, aribosomal binding site for effective translation. Additional elementsmay include terminator, signal, and upstream activating sequences.

The yeast cells are grown as described by Sherman in “Guide to yeastgenetics and molecular biology”, Guthrie and Fink, eds., Methods inEnzymology, Academic Press, Inc., Vol. 194, 3-21 [1991].

The baculovirus-insect cell vector system can also be used for theproduction of the SRTA-70 proteins of the present invention (for reviewsee Luclow and Summers, Bio Technology 6, 47-55 [1988]). The SRTA-70proteins produced in insect cells infected with recombinant baculoviruscan undergo post-translational processing including but not limited toN-glycosylation (Smith et al., Proc. Nat. Scad. Sci. USA 82, 8404-8408)and O-glycosylation (Thomsen et al., 12. International HerpesvirusWorkshop, University of Philadelphia, Pa.).

Mammalian cells can also be used as hosts for the recombinant productionof SRTA-70 proteins. Suitable mammalian host cells include but are notlimited to human Hela, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,CV1 African green monkey kidney cells, quail QC1-3 cells, Chinesehamster ovary (CHO) cells, mouse L cells and the COS cell lines.

Expression vectors suitable for use in mammalian host cells include butare not limited to pBC12MI, pBC12BI, pSV2dhFr, p91023(B), pcDNA3, pcDV1,pRSVcat, pGA291, pGA293, pGA296, pBC12/HIV/IL-2 and PGA300. Such vectorsare preferably introduced into suitable mammalian host cells bytransfection.

Usually, the mammalian host cells which contain a desired expressionvector are grown under conditions which are optimal for the growth ofthe mammalian host cells. A typical expression vector contains thepromoter element, which mediates the transcription of mRNA, the proteincoding sequence, and the signals required for efficient termination andpolyadenylation of the transcript. Additional elements may includeenhancers and intervening sequences bounded by spliced donor andacceptor sites.

Most of the vectors used for the transient expression of a given codingsequence carry the SV40 origin of replication, which allows them toreplicate to high copy numbers in cells (e.g. COS cells) thatconstitutively express the T antigen required to initiate viral DNAsynthesis. Transient expression is not limited to COS cells. Anymammalian cell line that can be transfected can be utilized for thispurpose. Elements that control a high efficient transcription includethe early or the late promoters from SV40 and the long terminal repeats(LTRs) from retroviruses, e.g. RSV, HIV, HTLVI. However, also cellularsignals can be used (e.g. human β-actin-promoter).

Alternatively stable cell lines carrying a gene of interest integratedinto the chromosome can be selected upon co-transfection with aselectable marker such as gpt, dhfr, neomycin or hygromycin.

Now, the transfected gene can be amplified to express large quantitiesof a foreign protein. The dihydrofolate reductase (DHFR) is a usefulmarker to develop lines of cells carrying more than 1000 copies of thegene of interest. The mammalian cells are grown in increasing amounts ofmethotrexate. Subsequently, when the methotrexate is withdrawn, celllines contain the amplified gene integrated into the chromosome.

Transgenic animal vector systems can also be used for the production ofSRTA-70 proteins of the present invention (for review see Pinkert,Transgenic animal technology: a laboratory handbook, Academic Press, SanDiego [1993]). Using specific signal sequences the desired SRTA-70protein can also be secreted into the milk of the animal (for examplessee Drohan et al., J. Cell. Biochemistry 17a, 38-38 [1993]; Lee et al.,Appl. Biochem. Biotechnol. 56, 211-222 [1996]) thus allowing the use ofthe milk as a source for SRTA-70 protein.

For the isolation of small amounts of SRTA-70 proteins expressed inprokaryotic host cells for analytical purposes, e.g., for polyacrylamidegel electrophoresis, the host cells can be disrupted by treatment with adetergent, e.g., sodium dodecyl sulphate (SDS). Larger quantities ofSRTA-70 protein can be obtained by mechanical (Charm et al., Meth.Enzymol. 22, 476-556 [1971]), enzymatic (lysozyme treatment) or chemical(detergent treatment, urea or guanidinium hydrochloride treatment, etc.)treatments followed by use of known methods, e.g., by centrifugation atdifferent gravities, precipitation with ammonium sulphate, dialysis (atnormal pressure or at reduced pressure), preparative isoelectricfocusing, preparative gel electrophoresis or by various chromatographicmethods such as gel filtration, high performance liquid chromatography(HPLC), ion exchange chromatography, reverse phase chromatography andaffinity chromatography (e.g., on Sepharose® Blue CL-6B).

Preferably, the SRTA-70 proteins expressed in prokaryotic host cells areobtained after Ni-Agarose affinity chromatography followed by gelfiltration.

The SRTA-70 proteins expressed in mammalian host cells or in thebaculovirus-insect cell vector system can be isolated from the host cellmedium using standard protein purification methods.

The SRTA-70 proteins can be used as therapeutically active agents inimmune response modulation, specifically, in augmentation andsuppression of the immune system.

Furthermore, the SRTA-70 proteins can be used as mediators ofprotein-protein interactions to retrieve other proteins involved in DNArecombination and repair, especially class switch recombination, andother metabolic processes. SRTA-70 proteins can serve as hooks to pullother relevant proteins out of cell extracts, and allow cloning therespective genes. SRTA-70 proteins can also be used for identificationof compounds inhibiting or boosting the function of SRTA-70 proteins andproteins and nucleic acids interacting with SRTA-70 proteins (agonistsor antagonists).

Antibodies can also be raised against the SRTA-70 proteins of thepresent invention. These antibodies can be used in a well-known mannerfor diagnostic or therapeutic purposes as well as for localisation andpurification purposes. Such antibodies can be produced by injecting amammalian or avian animal with a sufficient amount of a vaccineformulation comprising a SRTA-70 protein of the present invention and acompatible pharmaceutical carrier to elicit the production of antibodiesagainst said receptor. The appropriate amount of the SRTA-70 proteinswhich would be required would be known to one of skill in the art orcould be determined by routine experimentation. SRTA-70 specificantibodies may also be selected from phage, viral, or bacterial antibodylibraries. As used in connection with this invention the term“pharmaceutical carrier” can mean either the standard compositions whichare suitable for human administration or the typical adjuvants employedin animal vaccinations.

Suitable adjuvants for the vaccination of animals include but are notlimited to Freund's complete or incomplete adjuvant (not suitable forhuman or livestock use). Adjuvant 65 (containing peanut oil, mannidemonooleate, aluminum phosphate and alum; surfactants such ashexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N₁-N-dioctadecyl-N′-N-bis(2-hydroxyethylpropanediamine),methoxyhexy-decylglycerol, and pluronic polyols; polyanions such aspyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptidessuch as muramyl dipeptide, dimentylglycine, tuftsin; and oil emulsions.The SRTA-70 proteins could also be administered following incorporationinto liposomes or other microcarriers, or after conjugation topolysaccharides, other proteins or other polymers or in combination withQuil-A to form “Iscoms” (immuno-stimulating complexes) (Morein et al.,Nature 308, 457 [1984]).

Typically, the initial vaccination is followed some weeks later by oneor more “booster” vaccinations, the net effect of which is theproduction of high titers of antibodies against the SRTA-70 proteinswhich can be harvested in the usual way.

Another method consists in using the well-known Koehler and Milsteintechnique for producing monoclonal antibodies. In order to find outdifferent monoclonal antibodies which are directed against the sameantigen but against different epitopes, the method of Stähli et al. (J.of Immunological Methods 21, 297-304 [1980]) can be used.

The antibodies against the SRTA-70 proteins are useful for determinationof the expression (over- and underexpression) of SRTA-70 protein. As setforth below altered features of SRTA-70 expression may lead to cancerand allergy.

Various methods which are generally known can be employed in thedetermination of SRTA-70 protein.

In one such procedure known amounts of a sample to be assayed,radio-labeled SRTA-70 protein and unlabeled SRTA-70 protein are mixedtogether and allowed to stand. The antibody/antigen complex is separatedfrom the unbound reagents by procedure known in the art, e.g., bytreatment with ammonium sulphate, polyethylene glycol, second antibodyeither in access or bound to an insoluble support, dextran-coatedcharcoal and the like. The concentration of the labeled SRTA-70 proteinis determined in either the bound or unbound phase and the SRTA-70content of the sample can then be determined by comparing the level oflabeled component observed to a standard curve in a manner known per se.

Another suitable method is the “Double-Antibody-Sandwich-Assay”.According to this assay the sample to be tested is treated with twodifferent antibodies. One of these antibodies is labeled and the otheris coated on a solid phase.

Suitable solid phases are organic and inorganic polymers [amylases,dextrans, natural or modified celluloses, polyacrylamides, agaroses,magnetite, porous glass powder, polyvinylidene fluoride (Kynar) andlatex], the inner wall of test vessels (test tube, titer plates orcuvettes of glass or artificial material) as well as the surface ofsolid bodies (rods of glass and artificial material, rods with terminalthickening, rods with terminal lobes or lamellae). Spheres of glass andartificial material are especially suitable solid phase carriers.

Suitable labels are enzymes, e.g., peroxidase, radio-labels orfluorescence-labels.

Different antibodies can, e.g., be achieved by immunizing differentanimals, e.g., sheep and rabbits.

The methods for the determination of SRTA-70 protein as described abovecan be conducted in suitable test kits comprising in a containerantibodies against SRTA-70 protein elicited by a SRTA-70 protein of thepresent invention.

The isolated DNA sequences encoding SRTA-70 proteins are useful to makeprobes for assaying the status of the natural SRTA-70 gene (i.e.mutations, deletions, rearrangements, amplifications etc.), or itsexpression (over- and underexpression). This could be relevant to classswitch recombination, DNA recombination and repair in general, and otherprocesses. There might be diseases, that are linked to altered featuresof SRTA-70, for example, DNA repair deficiencies leading to cancer;class switch aberrations or redirections towards allergy-causing IgEexpression, and general B lymphocyte hypo- or hyperactivity.

The isolated DNA sequences encoding SRTA-70 proteins can also be used togenerate antisense RNA to alter expression of the endogeneous SRTA-70gene.

The isolated DNA sequences encoding SRTA-70 proteins, cloned intoappropriate vectors, can be used for overexpression of the gene intarget cells. This could create cellular models for the effect ofaltered SRTA-70 expression on class switch recombination, DNArecombination and repair, related processes, and general B cellfunction. This could allow to search for compounds which (counter-)regulate SRTA-70 protein expression.

Finally, the isolated DNA sequences encoding SRTA-70 proteins can beused to generate knockout mice. Such mice may have altered class switchrecombination, and/or altered DNA recombination and repair processes ingeneral. Such mice could be used as models for recombination-relateddiseases (cancer, allergies etc.), as well as for immune disordersrelated to B cell function, and as models for the respective therapeutictrials.

It has also been discovered that protein B23 has DNA recombinationfunctions. Thus, the present invention provides in addition the use ofprotein B23 as a mediator of protein-protein interactions to retrieveother proteins involved in DNA recombination and repair, especiallyclass switch recombination, and other DNA metabolic proceses. ProteinB23 could serve as a hook to pull other relevant proteins out of cellextracts. and allow cloning the respective genes. Protein B23 could alsobe used for screening of compounds inhibiting or boosting the functionof B23 (agonists or antagonists) and proteins and nucleic acidsinteracting with B23.

Isolated DNA sequences encoding protein B23 are useful to make probesfor assaying the status of the natural B23 gene (i.e. mutations,deletions, rearrangements, amplifications etc.), or its expression(over- and under-expression). This could be relevant to class switchrecombination, DNA recombination and repair in general, and otherprocesses. There might be diseases, that are linked to altered featuresof B23 (for example DNA repair deficiencies leading to cancer; classswitch aberrations or redirections towards allergy-causing IgEexpression).

Further it has been discovered that protein PARP interacts with proteinsB23 and SRTA-70. Thus, the present invention provides further the use ofprotein PARP as a regulator of the recombinative (DNA metabolic)functions of B23 and SRTA-70 and thereby involvement of PARP throughinteraction with SRTA-70 and B23 in class switch recombination; DNArepair and recombination, and related processes.

Having now generally described this invention, the same will becomebetter understood by reference to the specific examples, which areincluded herein for purpose of illustration only and are not intended tobe limiting unless otherwise specified, in connection with the followingfigures:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the DNA transfer assay. Thedouble-stranded (ds) M13 DNA contains Sγ and is digoxigenin-labeled; thepSP plasmid contains Sγ and is ³H labeled. The two substrates arecoincubated with a nuclear extract, e.g., from switching B cells. DNAtransfer from the Sγ plasmid to the Sμ plasmid results in plasmidscontaining both ³H and digoxigenin label. These plasmids can beprecipitated by a bead-bound antibody to digoxigenin, washed, and theirradioactivity measured in the scintillation counter.

FIG. 2 shows DNA transfer activity in extracts and extract fractionsfrom switching and non-switching splenic cell populations. (A) Nuclearextract (Fraction I; 850 ng/reaction) and SRTA Fraction II (80ng/reaction) tested with DNA substrates containing or lacking S-regions.(B) SRTA Fraction IV (1 ng/reaction) tested in two independentexperiments (stippled and black boxes) with DNA substrate combinationscontaining either both S regions, one S region (pSP-Sγ) and an unrelatedDNA (M13 RF, SV40, or ΦX174 RF), or no S regions (pSP+M13 RF) asindicated.

FIG. 3 shows analysis of switch recombination products. (A) PCR productswere analyzed by Southern blotting and hybridization with either an Sμor an Sγ probe as indicated. −T, without DNA templates; −P, no SRTAprotein added; +P, complete reaction; −γ, Sγ substrate omitted from therecombination reaction. X Sγ, hybridized with Sγ, X Sμ, hybridized withSμ. (B) Southern blot analysis of individually cloned PCR fragmentsobtained from DNA transfer reactions. M, size marker; γ and μ, plasmidscontaining Sγ and Sμ, respectively. X Sγ, hybridized with Sγ(3.7 kbEcoRI/HindIII fragment); X Sμ, hybridized with Sμ (1.3 kb HindIIIfragment).

FIG. 4 shows the DNA sequence of the SRTA-70 gene (SEQ ID No:2) and theamino acid sequence of the SRTA-70 protein (SEQ ID No:1). Shaded regionsindicate nuclear localization signals.

FIG. 5 shows protein interaction between B23, PARP, and SRTA-70. (A)Purification of overexpressed SRTA-70. His-tagged SRTA-70 (cDNA clonedinto pQE-30; Quiagen Inc.) was isolated from IPTG-induced E.coli,purified on a Ni-agarose column (Fr. I), followed by a Superdex 200 gelfiltration column (Fr. II). Un=uninduced, I=induced E.coli cell lysates(B and C). Protein fractions were eluted from the SRTA-70 affinitycolumn at the indicated ammonium sulfate concentrations and probed inWestern blots with (B) anti PARP antibody (Anwar Inc.), or (C) anti B23antibody (Chan et al., J. Biol. Chem. 261, 14335 [1986]). Numbers at thetop refer to mM ammonium sulfate used for elution.

FIG. 6 shows DNA dynamic activities of B23 protein. (A) Pairing ofcomplementary DNA single-strands. A heat-denatured 422 bp, ³²Pend-labeled DNA fragment was incubated with various amounts of B23protein, or E. coli RecA protein, or without protein (−), and in thepresence or absence of ATP or MgCl₂ as indicated. The assay wasperformed as described in EMBO J. 15, 4061-4068 [1996]. ss,single-strand substrates, ds, double-strand reannealed product. (B) and(C) Formation of joined molecules in a D-loop assay as described. 1,product, 2, unspecific ds substrate band, 3, ss substrate. (B) Linear orsupercoiled pSP-Sγ plasmid DNA and the ³²P labeled ss oligonucleotide(Sγ, 49 nt) with or without B23 protein under various conditions asindicated. (C) The Sγ oligonucleotide was incubated with (50 or 100 ng)or without (−) B23 and either pSP-Sγ, pSP-Sμ or pSP plasmid DNA.

FIG. 7 shows endonuclease activity in SRTA. The ds M13-Sμ DNA (50 ng)was incubated for the times indicated with or without SRTA protein inthe standard DNA transfer reaction buffer. After SDS/proteinase Ktreatment, products were analysed by gel electrophoresis (0.5% agarose,0.5×TBE), Southern blotting, and hybridisation with ³²P-labeled M13-SμDNA. Cleavage products (A, B) appear in the lower part of the gel.

FIG. 8 shows activated B lymphocyte-specific expression of SRTA-70protein. Nuclear extract protein from the various tissues and cellsindicated was analysed by SDS-PAGE and immuno blotting using polyclonalrabbit anti-SRTA-70 antibodies (affinity-purified on a SRTA-70 affinitycolumn).

LPS, lipopolysaccharide-stimulated cells

ConA, Concanavalin A stimulated cells

CD3^(−/−), spleen cells from a CD3^(−/−) mouse (no T-cells)

memCμ^(−/−), spleen cells from a membrane Cμ-deficient mouse (noB-cells)

wt, wildtype.

EXAMPLE 1 Purification of SRTA-70

Plasmid pSP72 containing 2.1 kbp Sγ2b sequences was labeled with³H-thymidine, and double-stranded (ds) M13 containing 1.3 kbp Sμsequences was labeled with a small number of digoxigenin ligands. An Sregion preferring recombination activity should catalyse the formationof recombinant DNA molecules, of which one example is shown in FIG. 1,containing both labels. The amount of such molecules can be measured bycounting ³H in plasmid DNA that has been immunoprecipitated by ananti-digoxigenin antibody.

Since splenic B cell cultures contain many different cell types,including a large portion of non-B lymphocytes and non-lymphocyticcells, lipopolysaccharide (LPS) stimulated (i.e. switching) B cellblasts were separated from the non-switching cells according to size bycell elutriation (Sanderson et al., Anal. Biochem. 71, 615-622 [1976]).Nuclear extracts were prepared as described (Jessberger and Berg, Mol.Cel.. Biol. 11, 445-457 [1991]), from 1×10⁸ to 8×10⁸ LPS (50 μg/ml)blasts (0.7 mg nuclear protein/10⁸ cells). Nuclear extracts were testedfor DNA transfer activities as described (Jessberger and Berg, supra).Input ³H radioactivity was between 150000 and 350000 cpm and the samefor each experimental series. The Sμ substrate consisted of an M13 dsDNA carrying a 1.3 kbp HindIII Sμ fragment (DePinho et al., Mol. Cell.Biol. 4, 2905-291 [1984]), and was labeled with digoxigenin (Jessbergerand Berg, supra). The Sγ plasmid consists of pSP72 containing a 3.7 kbEco RI-HindIII Sγ2b fragment (De Pinho et al., Mol. Cell. Biol. 4,2905-2912 [1984]); it was internally labeled with ³H-thymidine(Jessberger and Berg, supra). For the standard DNA transfer assay 0.18μg of the ³H labeled DNA (e.g. pSP-Sγ) and 0.02 μg of the dig-labeledDNA (e.g. M13-Sμ) were coincubated with varying amounts of protein in 50μl containing 3 mM MgCl₂, 30 mM EPPS, pH 7.4, 1 mM DTT, less than 50 mMammonium sulfate, and 1 mM ATP. After 6 min the reaction was terminatedby the addition of EDTA to 75 mM and SDS to 0.02% and heated to 65° C.for 20 min. The reaction mixture was extracted with phenol-chloroform(1:10) and incubated with anti digoxigenin beads (Jessberger and Berg,supra). The beads were collected on glass wool, washed with PBS-0.05%Tween-20, and the radioactivity of both the bead-bound and unbound DNA(together accounting for the total radioactivity) counted separately ina scintillation counter.

Despite the presence of general DNA transfer activities, the crudenuclear extracts from LPS blasts recombined S region substrates (SRS)two- to threefold better than non-S region substrates (NSRS), and thispreference was not seen in extracts prepared in parallel from thenon-switching splenic cell pool (FIG. 2A). This indicates induction of anew activity in switching cells.

Fraction I (2 mg protein) was loaded onto a Superdex 200 FPLC gelfiltration column (Pharmacia) and fractionated at a flow rate of 1ml/min in buffer E (5 mM KCl, 5 mM MgCl₂, 2 mM DTT, 0.2 mM EDTA, 15 mMTris-HCl, pH 7.5 at 4° C., and 1 mM PMSF, 10 mM Na₂S₂O₅, 1 μg/mlaprotinin, 0.5 μg/ml TLCK, 0.7 μg/ml pepstatin A) containing 80 mMammonium sulfate. 1.4 ml fractions were collected. Active fractionseluting around 57-61% column volume were pooled (5.6 ml, 0.6 mg protein,Fraction II), diluted 1:4 with buffer E and loaded at 1 ml/min onto a 1ml Macro S cation exchange FPLC column (BioRad). After washing thecolumn with 20 column volumes buffer E-20 (E plus 20 mM ammoniumsulfate), the proteins were eluted at a 1 ml/min flow rate with agradient from 20 to 600 mM ammonium sulfate in buffer E in 1.2 mlfractions. The switch-specific activity (2.4 ml, 0.011 mg protein, Fr.III) eluted in two fractions at around 280 mM ammonium sulfate. Forfurther purification, Fraction III was diluted 1:2 with buffer E andloaded at 0.3 ml/min onto a 1 ml Blue-Sepharose (HiTrap, Pharmacia) FPLCcolumn (pre-equilibrated in E-140). Elution was with a linear gradientfrom 0 to 1000 mM ammonium sulfate in buffer E, and the activity elutedbetween 740 and 810 mM (0.6 ml, 0.0006 mg protein, Fr. IV). On ice or at−70° C. the active fractions were stable for a short period only; frozenin liquid nitrogen, samples remained active for at least several weeks.

Fraction II showed an about 4 fold preference for SRS and was completelyinactive if isolated from non-switching cells (FIG. 2A). The S regionspecific activity eluted at about 280 mM ammonium sulfate showed anine-fold preference. Fraction IV the preference for S region substratesover non-S region substrated was about tenfold (FIG. 2B), with thenon-S-substrates reaction yielding almost background levels of activity.With these purification steps the specific activity for DNA transferbetween the S-substrates increased more than thousand-fold. Reactionsthat included one S region and an unrelated DNA like M13, SV40 or ΦX174DNA as the second partner yielded low activity (FIG. 2B). Homologous DNAsubstrates (5.7 kbp homology) recombined with lower efficiency (app.60%) than combinations of two SRS, as did substrates which sharedlimited homology (ca. 2 kbp stretch of homology; 45% efficiency) betweenthem. Homology, therefore, is not sufficient to drive the reaction.

As maximum product formation by Fraction IV (1 ng) occurred after 6 minincubation at 37° C., and at 3 mM MgCl₂, these conditions were definedas standard conditions. Omission of the four dNTPs did not affect thereaction much (1.97%cpm versus 1.75%cpm of a standard reaction). Incontrast, lack of ATP rendered the reaction 88% less efficient. Whenboth the ATP and the four dNTPs were omitted, product formation wasdecreased to a similar degree (0.2%cpm versus 1.75%cpm of a standardreaction; and 0.2%cpm versus 1.97%cpm of the reaction lacking dNTPs).The dependence on ATP indicates energy cofactor requirement, but DNAsynthesis seems not to be necessary, and no DNA polymerase activity wasdetected in Fractions III and IV; the fractions also lackedtopoisomerase I and II, and DNA helicase activities.

Since the DNA was treated with SDS/EDTA and phenol, the linkage of thetwo substrates is considered stable and independent of the continuouspresence of protein. More than 80% of the transfer products are heatstable (20 min at 85° C.).

In a next step, the structure of the recombination products was analyzedby PCR. Due to the imprecision of the switch recombination reaction andthe possibility of multiple rearrangement events this analysis requiredspecial provisions. Thus, only the affinity-bead bound DNA was amplifiedwhich includes the digoxigenin-labeled substrate (M13-Sμ) and the DNAtransfer products, but not the other substrate (pSP-S) DNA. The PCRprimers were specific for S and Sμ and, thereby, diagnostic forjunctions between two S regions. PCR reaction: Sμ-primer5′-GATGGGTGGGCTTCTCTGAGCG (SEQ ID NO:3) (5′ region of Sμ, bp. No.67-88); S-primer 5′GTATTAGGGACCAGTCCTATCAG (SEQ ID NO:4) (middle of S,bp. No. 1076-1098); 1 min at 95° C. and 25 cycles of 20 s 95° C., 20 sat 50° C. and 1.1 min at 72° C. Amplification products were analyzed bySouthern blot hybridization with either an S or an Sμ probe (FIG. 3A).As controls, the active protein (Fr. IV) or the DNA templates wereomitted from the recombination reaction, or only one DNA substrate (Sμ)was used. Only when the DNA transfer reaction contained both DNAsubstrates together with the protein fraction did the products hybridizeto both Sμ and S region probes (FIG. 3A). As expected from theimprecision of switch recombination, the observed products wereheterogenous, though not entirely random, as the DNA transfer reactionand the PCR design may select for subsets of products. They alsoincluded Sμ-Sμ junctions. PCR products shown in FIG. 3A were subclonedinto an unrelated plasmid vector and colony hybridization screens ofcloned DNA were performed with either the S or Sμ probe. About 20% ofthe clones contained DNA hybridizing with both probes. The plasmidspurified from the clones were linearized and Southern-hybridized with Sand Sμ probes. As shown in FIG. 3B, seven of nine clones shown containedS and Sμ sequences of various sizes in the same molecule.

On silver stained SDS polyacrylamide gels there were 10 polypeptidesleft in Fraction III, and 6 polypeptides in Fraction IV. The prominentspecies in Fraction IV have approximate molecular weights of 38, 50, 70,75, 115 and 160 kDa, respectively. The 38, 70, and 115 kDa proteins weregel-eluted and partial amino acid sequences of them were determined. Twotryptic peptides of the 38 kDa protein were TVSLGAG (SEQ ID NO: 5) andFINYVKI (SEQ ID NO: 6); of the 115 kDa protein TLGDFLAEYAK (SEQ ID NO:7) and TTNFAGILSQG (SEQ ID NO: 8); and the N-terminal sequence of the 70kDa protein was MRGLKDELLKAIWHAFTALDLDRS (SEQ ID NO: 9). The 38 kDaprotein was identified as B23 (nucleophosmin; Chan et al., J. Biol.Chem. 261, 14335-14341 [1986]) and the 115 kDa protein aspoly(ADP-ribose) polymerase (PARP; de Murcia and de Murcia, TrendsBiochem. Sci. 19, 172-176 [1994]). The identifications were confirmed byWestern blotting experiments. The complete sequence of the 70 kDaprotein, named SRTA-70, is shown in FIG. 4. It does not belong to aknown protein family and contains nuclear localization signals, apossible coiled-coil region between amino acids 320 and 450, a potentialO-glycosylation site at amino acids 314/315, and a continuoushydrophilic region near its C-terminus.

EXAMPLE 2 Cloning of the DNA Sequence Encoding SRTA-70 and Expressionand Purification of SRTA-70

The N-terminal amino acid sequence of SRTA-70 described in Example 1 wasused to synthesize the oligonucleotides A and B, set forth below. Theseoligonucleotides correspond to either end of the N-terminal amino acidsequence of SRTA-70 and allow the generation of a RT-PCR product (72 bp)covering the entire N-terminal amino acid sequence of SRTA-70. From this72 bp PCR product, an authentic 23 nt oligonucleotide was derived (see Cbelow), which corresponds to the middle region of the N-terminal aminoacid sequence of SRTA-70. The oligonucleotide C was then used as a³²P-labeled hybridization probe to screen a cDNA library (obtained fromStratagene Inc., mouse spleen cDNA library from 8-12 weeks old C57BL/6female mice; Lambda ZAPII Vector; Catalog No. 936308). One positiveclone was purified by three rounds of plaque purification (replating andhybridisation with C). It contained a 2.8 kbp insert spanning the entireSRTA-70 cDNA.

A: 5′-ATG MGN GGN YTN AAA GAC GA (SEQ ID NO: 10)

B: 5′-GT RAA NGC ATG CCA GAT (SEQ ID NO: 11)

C: 5′-GAA CTG CTC AAA GCC ATH TGC CA (SEQ ID NO: 12)

M=A or C, Y=C or T, N=A, T, G or C, R=A or G and H=A, C or T.

The SRTA-70 cDNA contained in the plasmid Bluescript in the Lambdavector was excised from the Lambda ZAPII Vector using the helper phageassisted excisison procedure given by Stratagene Inc. and using thematerial provided by Stratagene. The Bluescript-SRTA-70 clone DNA wasthen used as starting material for subcloning the cDNA into the pQE-30vector (Quiagen Inc.) for expression of the his-tagged protein in E.coli. The cDNA was inserted into the Bam HI and EcoRV sites of thepQE-30 vector, transfected into the M15 E. coli host strain (QuiagenInc.), and expression of the protein induced in positive clones by IPTG(1 mM) addition to the medium. Expression was monitored by SDS-PAGEanalysis of E. coli cell extracts and Comassie staining. Clones thatshowed a strongly induced 70 kDa protein band were used for furtheranalysis and larger scale expression. From these, cell lysates wereprepared according to the following procedure:

cells were pelleted by centrifugation from the medium after 2 hinduction in the presence of IPTG, and resuspended in ice-cold lysisbuffer (50 mM Tris.HCl, pH 8.0, 300 mM NaCl, containing 1 tabletcomplete protease inhibitor unit from Boehringer Mannheim Inc., Cat. No.1836153). Lysozyme was added to 2 mg/ml, and the cells incubated for 30min. on ice. Imidazole and PMSF were added to 1 mM each, and the cellsuspension was sonicated 5 times for 1 min until a viscous solution wasgenerated. The insoluble fraction was removed by centrifugation (SS-34rotor, Sorvall, 18000 rpm, 4° C., 20 min), and the clear supernatant,containing the SRTA-70 protein, collected.

This solution was then applied to a Ni-Agarose column for affinitychromatographic purification of the his-tagged SRTA-70, as suggested bythe manufacturer (Quiagen Inc.). The column was washed with 10 volumeslysis buffer containing 1 mM Imidazole and then stepwise eluted with 20,40, 80, 120, 200 mM Imidazole in the same buffer. The SRTA-70 proteineluted mainly in the 80 mM Imidazole step, as seen by SDS-PAGE analysisof the fractions. For further purification, this fraction was loaded ona Superdex 75 FPLC gel filtration column (Pharmacia), developed in abuffer containing 50 mM ammonium sulfate, 10% glycerol, proteaseinhibitors as above, 1 mM EDTA. The fractions containing SRTA-70 werefrozen in aliquots in liquid niotrogen and stored at −70° C.

Expression and purification of SRTA-70 as a his-tagged molecule in E.coli yielded a >95% pure preparation (FIG. 5A, Frct. II). Thispreparation was used as an affinity-tag, bound to sepharose beads, forproteins contained in the nuclear extract from switching B cells. Thebound material was stepwise eluted with 80, 120, 300, 600 and 1200 mMammonium sulfate, and the fractions were analysed by SDS-PAGE andWestern blotting. The 300, 600 and 1200 mM fractions contained only veryfew polypeptides, as judged from silver staind gels. Western blottingrevealed PARP peaking in the 300 (FIG. 5B) and B23 peaking in the 300and 600 mM fractions (FIG. 5C), corresponding to 600 and 1200 mM ionicstrength, respectively, of a mono-valent salt. This indicated highaffinity protein-protein interactions between B23, PARP, and SRTA-70.

EXAMPLE 3 Expression and Purification of Human SRTA-70

Human cDNA for SRTA-70 is cloned by using moderately degenerate PCRprimer derived from the mouse cDNA sequence in a standard RT-PCR scheme(RT=Reverse Transcription). The human cDNA is alternatively cloned bythe use of the one EST existing in the data bank (Accession No. W 39285)as a probe for screening human cDNA libraries. This EST is 89%homologous to the 3′ end of the mouse SRTA-70.

EXAMPLE 4 Activities of B23 and PARP

The ability of SRTA-70 and B23 to promote DNA pairing reactions wastested. Both proteins were overexpressed and purified from E. coli (FIG.5A; Wang et al., J. Biol. Chem. 269, 30994-30998 [1994]). SRTA-70 wasnot active in the pairing assay, but B23 was at least as efficient asthe E. coli RecA protein: 15 ng of B23 yielded as much double stranded(ds) DNA as 24 ng of RecA (FIG. 6A).

For a more complex three-strand pairing reaction-probably closer to theswitch reaction—the formation of joined molecules generated by invasionof ss DNA into a ds target, the so-called D-loop formation(Kowalzykowski and Eggleston, Ann. Rev. Biochem. 63, 991-1043 [1994];Beattie et al., J. Mol. Biol. 116, 783-803 [1977]) was tested.

25 fmoles predominantly supercoiled plasmid DNA and 0.6 pmoles (1.0 ng)5′-32P labeled, 49 nt single-stranded S oligonucleotide (5′ GGGACCAGTCCTAGCAGCTGTGGGGGAGCTGGGGAAGGTGGGAGTGTGA) (SEQ ID NO: 13) wereincubated together with protein for 30 min. at 37° C. in the standardDNA transfer reaction buffer. Reactions were stopped by addition of SDSto 0.1% and 4 μg Proteinase K and further incubation at 37° C. for 45min. Products were analysed in 0.6% agarose gels containing 3 mMMg-acetate in the TAE buffer system. Gels were run at 0.8 V/cm for 24 hat 4° C., stained with ethidium bromide, photographed, dried and exposedfor autoradiography for 2-16 h.

As seen in the four left lanes in FIG. 6B, linear plasmid DNA is not asubstrate in the D loop reaction with 5′-³²P labeled single-strandoligonucleotide, as it is not in the reaction mediated by E. coli RecA(Beattie et al., supra). There is known unspecific, i.e., proteinindependent pairing, which probably is due to annealing of theoligonucleotide to partially and irreversibly denatured supercoiled DNA(band 2 in all lanes of FIG. 6B). During the specific reaction, however,the supercoiled substrate DNA is partially relaxed to the circular form,which constitutes the product (band 1). As shown in FIG. 6B, B23transferred the 49 nt Sγ oligonucleotide into the predominantlysupercoiled pSP-Sγ to produce band 1 in lanes 5, 6, 8, 9, and 10(SRTA-70 was not active in this reaction). The activity of B23 isinhibited by the presence of 1 mM ATP (lane 11), but the inhibition canbe overcome by inclusion of PARP in the reaction (lanes 6, 8, 9). PARPitself, however, is inactive in D-loop formation (FIG. 6B, lane 7). Itis known that B23 can be modified by PARP and that it binds PAR polymers(Ramsamooi et al., Rad. Res. 143, 158-164 [1995]. The modification byPARP shown here does not depend on the presence of NAD (FIG. 6B, lanes5, 8, 10) and thus could either be caused by direct protein-proteininteractions, or by PAR polymers, present in the PARP preparation. ATPmay inhibit B23 by occupying the polymer binding site, and may becompeted out by the polymers. This mechanism might constitute a novelway to regulate B23 DNA dynamic activity. Indeed, in the SRTA fractions,about half of B23 was found in Western blotting experiments with antiPAR antibodies to contain the polymers.

The homology between the 49 nt oligonucleotide and the pSP-Sγ target isto the best 92% over a 48 bp stretch. Since there exist patches ofhomologies between different S regions, it was tested for joinedmolecule formation with the Sγ oligonucleotide and a pSP-Sμ ds targetDNA (FIG. 6C). The maximal homology here is 75% in a 48 bp stretch or90% in a 11 bp stretch. Though not as efficient as the Sγ-Sγ pairing(right 3 lanes), B23 clearly was able to produce joined molecules withthe two different S regions (middle 3 lanes). No joined molecules wereobtained with the pSP plasmid DNA as target (left 3 lanes), althoughonly slightly lower levels of homology exist (70-80% in stretches of15-20 bp). Thus, small patches of homologies in the S regions maysupport pairing of different S regions but other sequence or structuralelements, or a minimal length of stretches of homology as present in Sregions are necessary to form stable products. B23 was also active inanother three-strand reaction: DNA strand exchange between a linear,3′-P32 labeled, 422 pb double-strand M13 DNA fragment and thesingle-strand, circular M13 phage DNA.

Although B23 can provide important DNA recombinative functions, it isnot sufficient for the complete DNA transfer reaction, which requiresthe SRTA fraction. Among additional activities may be an endonucleolyticactivity that initiates DNA transfer between two covalently closedcircular DNA molecules. Such a requirement in switch recombination canalso be deduced from the loop-excision model (Jäck et al., Proc.Natl.,Acad. Sci. USA 85, 1581-1585 [1988]). Thus the SRTA was analysed for thepresence of nuclease activities. These included 5′-3′ ds exonuclease,3′-5′ ds exonuclease, ss endonuclease, ds endonuclease on linear DNAfragments, and ds endonuclease on predominantly supercoiled plasmid orphagemid DNA molecules. The only endonuclease detected cleavedsupercoiled plasmid or phagemid DNA (FIG. 7). In Southern blotting ofthe product DNAs, the cleavage product appeared after 1-2 min incubationand increased thereafter. None of the other nuclease activitiescopurified with the SRTA. This DNA double-strand specific endonucleasedid not depend on ATP or NAD, and is not a topoisomerase II activity, asthis was absent from the preparation. The endonuclease was not specificfor plasmids containing S regions. Secondary structures present in manyplasmids and phagemids, and known to be formed by S regions may serve ascleavage signals. The cleavage products generated by this endonucleasemight activate PARP, which depends on binding to DNA nicks ordouble-strand breaks (deMurcia and deMurcia, supra).

EXAMPLE 5 Expression of SRTA-70 Protein

Expression of SRTA-70 protein was investigated using standardimmuno-blotting techniques and the antibodies mentioned above. A seriesof protein extracts from various tissues and either ConA- orLPS-stimulated spleen cells was probed (FIGS. 8A,B). High expression ofSRTA-70 protein was found only in activated B lymphocytes.

13 1 585 PRT Mus musculus 1 Met Arg Gly Leu Lys Asp Glu Leu Leu Lys AlaIle Trp His Ala Phe 1 5 10 15 Thr Ala Leu Asp Leu Asp Arg Ser Gly LysVal Ser Lys Ser Gln Leu 20 25 30 Lys Val Leu Ser His Asn Leu Cys Thr ValLeu Lys Val Pro His Asp 35 40 45 Pro Val Ala Leu Glu Glu His Phe Arg AspAsp Asp Glu Gly Pro Val 50 55 60 Ser Asn Gln Gly Tyr Met Pro Tyr Leu AsnLys Phe Ile Leu Glu Lys 65 70 75 80 Val Gln Asp Asn Phe Asp Lys Ile GluPhe Asn Arg Met Cys Trp Thr 85 90 95 Leu Cys Val Lys Lys Asn Leu Thr LysSer Pro Leu Leu Ile Thr Glu 100 105 110 Asp Asp Ala Phe Lys Val Trp ValIle Phe Asn Phe Leu Ser Glu Asp 115 120 125 Lys Tyr Pro Leu Ile Ile ValPro Glu Glu Ile Glu Tyr Leu Leu Lys 130 135 140 Lys Leu Thr Glu Ala MetGly Gly Gly Trp Gln Gln Glu Gln Phe Glu 145 150 155 160 His Tyr Lys IleAsn Phe Asp Asp Asn Lys Asp Gly Leu Ser Ala Trp 165 170 175 Glu Leu IleGlu Leu Ile Gly Asn Gly Gln Phe Ser Lys Gly Met Asp 180 185 190 Arg GlnThr Val Ser Met Ala Ile Asn Glu Val Phe Asn Glu Leu Ile 195 200 205 LeuAsp Val Leu Lys Gln Gly Tyr Met Met Lys Lys Gly His Lys Arg 210 215 220Lys Asn Trp Thr Glu Arg Trp Phe Val Leu Lys Pro Asn Ile Ile Ser 225 230235 240 Tyr Tyr Val Ser Glu Asp Leu Lys Asp Lys Lys Gly Asp Ile Leu Leu245 250 255 Asp Glu Asn Cys Cys Val Glu Ser Leu Pro Asp Lys Asp Gly LysLys 260 265 270 Cys Leu Phe Leu Ile Lys Cys Phe Asp Lys Thr Phe Glu IleSer Ala 275 280 285 Ser Asp Lys Lys Lys Lys Gln Glu Trp Ile Gln Ala IleTyr Ser Thr 290 295 300 Ile His Leu Leu Lys Leu Gly Ser Pro Pro Pro HisLys Glu Ala Arg 305 310 315 320 Gln Arg Arg Lys Glu Leu Arg Arg Lys LeuLeu Ala Glu Gln Glu Glu 325 330 335 Leu Glu Arg Gln Met Lys Glu Leu GlnAla Ala Asn Glu Asn Lys Gln 340 345 350 Gln Glu Leu Glu Ser Val Arg LysLys Leu Glu Glu Ala Ala Ser Arg 355 360 365 Ala Ala Asp Glu Glu Lys LysArg Leu Gln Thr Gln Val Glu Leu Gln 370 375 380 Thr Arg Phe Ser Thr GluLeu Glu Arg Glu Lys Leu Ile Arg Gln Gln 385 390 395 400 Met Glu Glu GlnVal Ala Gln Lys Ser Ser Glu Leu Glu Gln Tyr Leu 405 410 415 Gln Arg ValArg Glu Leu Glu Asp Met Tyr Leu Lys Leu Gln Glu Ala 420 425 430 Leu GluAsp Glu Arg Gln Ala Arg Gln Asp Glu Glu Thr Val Arg Lys 435 440 445 LeuGln Ala Arg Leu Leu Glu Glu Glu Ser Ser Lys Arg Ala Glu Leu 450 455 460Glu Lys Trp His Leu Glu Gln Gln Gln Ala Ile Gln Thr Thr Glu Ala 465 470475 480 Glu Lys Gln Glu Leu Glu Gln Gln Arg Val Met Lys Glu Gln Ala Leu485 490 495 Gln Glu Ala Met Ala Gln Leu Glu Gln Leu Glu Leu Glu Arg LysGln 500 505 510 Ala Leu Glu Gln Tyr Glu Gly Val Lys Lys Lys Leu Glu MetAla Thr 515 520 525 His Met Thr Lys Ser Trp Lys Asp Lys Val Ala His HisGlu Gly Leu 530 535 540 Ile Arg Leu Ile Glu Pro Gly Ser Lys Asn Pro HisLeu Ile Thr Asn 545 550 555 560 Trp Gly Pro Ala Ala Phe Thr Gln Ala GluLeu Glu Glu Arg Glu Lys 565 570 575 Ser Trp Lys Glu Lys Lys Thr Thr Glu580 585 2 1758 DNA Mus musculus 2 atgagggggt tgaaagacga actgctcaaagccatttggc acgccttcac cgcgctcgac 60 ctggaccgca gcggcaaggt ctccaagtcgcaactcaagg tcctttccca taacctgtgc 120 acggtgctga aggttccaca tgacccggttgcccttgagg agcactttag ggatgacgat 180 gaggggcctg tctccaatca gggctacatgccatatttaa acaagttcat tttggaaaag 240 gtccaagaca actttgacaa gattgaattcaatagaatgt gttggacact ttgtgtcaag 300 aaaaacctca caaagagtcc tctactcattacagaagatg atgcatttaa agtgtgggtc 360 attttcaact ttttgtcaga ggacaagtatccactaatta ttgtgccaga agagattgaa 420 tacctgctta agaagcttac agaagctatgggaggaggtt ggcaacaaga acaatttgaa 480 cattacaaaa taaactttga tgacaataaagatggccttt ctgcatggga acttattgag 540 ctaattggga atggacagtt tagcaagggcatggaccgtc agaccgtatc tatggccatt 600 aacgaagtct tcaatgagct tattttagatgtattgaagc agggttacat gatgaagaaa 660 ggtcacaaac ggaaaaactg gactgagcgctggtttgtat taaaacccaa cataatttcc 720 tactatgtga gcgaggatct gaaagataagaaaggagaca tcctgctgga tgaaaactgc 780 tgtgtggagt ctctgcctga caaagatggaaagaaatgtc tttttctaat aaaatgcttt 840 gataagacct ttgaaatcag tgcctcagataagaagaaga aacaagaatg gattcaggcc 900 atttactcca ccatccatct gttgaagctgggcagccccc caccacacaa ggaagcccgc 960 cagcgtcgga aagagctccg aaggaagctgctagccgagc aggaggagct ggagcggcag 1020 atgaaggaac tccaagccgc caatgaaaacaagcaacagg agctggaaag cgtgaggaag 1080 aaactggagg aagcagcctc tcgtgcggcagacgaggaaa agaaacgctt gcagactcag 1140 gtggagctac agaccaggtt cagcacggagctggagcggg agaagctgat cagacagcag 1200 atggaggagc aggttgccca gaagtcctccgaactggagc agtatctgca gcgagttcgg 1260 gagctggaag acatgtacct aaagctgcaggaggctcttg aggacgagag gcaggcccgg 1320 caggatgaag agactgtgcg caagcttcaggccaggttgc tggaggaaga gtcttctaag 1380 agggcagagc tggaaaagtg gcacctggagcagcagcagg ccattcagac aacagaggcg 1440 gagaagcagg agctggaaca gcagcgtgtcatgaaggagc aggcattgca ggaggccatg 1500 gcacagctgg aacagttgga gttggagcggaagcaggccc tggagcagta tgagggagtt 1560 aaaaagaagc tagagatggc aacacatatgaccaagagct ggaaggacaa agtggcccat 1620 catgagggat taatacgatt gatagaaccaggttccaaga accctcatct gatcaccaac 1680 tggggacccg cagcgttcac ccaggcagagctcgaggaga gagagaagag ctggaaagag 1740 aagaagacca cagagtga 1758 3 22 DNAArtificial Sequence primer 3 gatgggtggg cttctctgag cg 22 4 23 DNAArtificial Sequence primer 4 gtattaggga ccagtcctat cag 23 5 7 PRT Musmusculus 5 Thr Val Ser Leu Gly Ala Gly 1 5 6 7 PRT Mus musculus 6 PheIle Asn Tyr Val Lys Ile 1 5 7 11 PRT Mus musculus 7 Thr Leu Gly Asp PheLeu Ala Glu Tyr Ala Lys 1 5 10 8 11 PRT Mus musculus 8 Thr Thr Asn PheAla Gly Ile Leu Ser Gln Gly 1 5 10 9 24 PRT Mus musculus 9 Met Arg GlyLeu Lys Asp Glu Leu Leu Lys Ala Ile Trp His Ala Phe 1 5 10 15 Thr AlaLeu Asp Leu Asp Arg Ser 20 10 20 DNA Artificial Sequence SyntheticOligonucleotide 10 atgmgnggny tnaaagacga 20 11 17 DNA ArtificialSequence Synthetic Oligonucleotide 11 gtraangcat gccagat 17 12 23 DNAArtificial Sequence Synthetic Oligonucleotide 12 gaactgctca aagccathtgcca 23 13 49 DNA Artificial Sequence Synthetic Oligonucleotide 13gggaccagtc ctagcagctg tgggggagct ggggaaggtg ggagtgtga 49

What is claimed is:
 1. An isolated polynucleotide comprising a DNAsequence having greater than 95% sequence identity to the nucleotidesequence set forth in SEQ ID NO:02, wherein said polynucleotide encodesan S-Region Transfer Activity-70 (SRTA-70) protein, wherein said SRTA-70protein mediates recombination between immunoglobulin heavy chain switchregions.
 2. The polynucleotide according to claim 1 comprising thenucleotide sequence set forth in SEQ ID NO:
 2. 3. A vector comprisingthe polynucleotide as claimed in claim
 1. 4. The vector as claimed inclaim 3 wherein said vector is capable of directing expression inprokaryotic, yeast, plant, mammalian or insect host cells.
 5. Anisolated host cell comprising the vector as claimed in claim 3 whereinsaid host cell is selected from the group consisting of a prokaryotecell, a yeast cell, a plant cell, a mammalian cell and an insect cell.6. The host cell of claim 5 which is a prokaryote cell.
 7. A method forproducing an SRTA-70 protein comprising cultivating a host cell asclaimed in claim 5 in a suitable medium; and isolating said protein. 8.The polynucleotide of claim 1, wherein said SRTA-70 protein binds polyADP ribose polymerase and B23.
 9. An isolated SRTA-70 protein having anamino acid sequence greater than 95% sequence identity to the amino acidsequence set forth in SEQ ID NO:01, wherein said SRTA-70 proteinmediates recombination between immunoglobulin heavy chain switchregions.
 10. The protein according to claim 9, wherein said proteincomprises an amino acid sequence as set forth in SEQ ID No:
 1. 11. TheSRTA-70 protein of claim 9, wherein said SRTA-70 protein binds poly ADPribose polymerase and B23.